【作者】 彭倩宜；

【导师】 陶立坚； 艾宇航；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 目的：前B细胞集落增强因子(pre-B cell colony enhancing factor, PBEF)是联系炎症与胰岛素抵抗的桥梁,是研究和治疗炎症所致胰岛素抵抗的潜在靶点。然而PBEF与胰岛素通路是否存在相互作用及其机制一直存在很大争议。脂筏作为膜信号转导的重要平台,在胰岛素信号通路中发挥着重要作用。本研究首次从一个全新的角度——脂筏来探究PBEF与胰岛素受体(insulin receptor, IR)及胰岛素信号通路相互作用的机制,以期揭示炎症所致的胰岛素抵抗的新机制。方法：采用去垢剂及非去垢剂法从A549细胞系中提取脂筏,鉴定PBEF与IRβ在脂筏内外的分布。构建重组PBEF质粒及其突变质粒S199,提取PBEF及S199蛋白,研究PBEF对IRp在脂筏内外分布的影响。运用免疫共沉淀(immunoprecipitation, IP)研究PBEF、IRβ与Caveolin-1(Cav-1)的相互作用,及其与Cav-1磷酸化的关系。采用不同时间点研究PBEF对胰岛素诱导的Akt活化的影响。结果：PBEF在脂筏内外均有分布,而IRp在非去垢剂法中仅存在于脂筏。PBEF能诱导IRβ从脂筏转移至非脂筏,NAD也具有类似作用,但该作用不能完全被S199和FK866阻断。IRβ、PBEF与Cav-1三者相互结合。PBEF可导致非脂筏中与IRp结合的Cav-1磷酸化水平明显增加。给予胰岛素处理5min内加入PBEF,Akt的磷酸化水平较胰岛素单独作用时降低；而给予胰岛素处理5min后再加入PBEF,Akt的磷酸化水平部分恢复。结论：1、PBEF在脂筏内外均有分布。PBEF能诱导IRp从脂筏转移至非脂筏,该作用部分依赖其Nampt酶活性。2、IRβ、 PBEF与Cav-1三者相互结合。PBEF促进磷酸化Cav-1与IRp共同移向非脂筏,且该作用与其Nampt酶活性相关。3、PBEF能影响胰岛素诱导的Akt活化,且该作用具有时间依赖性。更多还原

【Abstract】 Purpose:Pre-B cell colony enhancing factor (PBEF) is a bridge between inflammation and insulin resistance, and a potential target for the research and treatment of inflammation-induced insulin resistance. However, the interaction between PBEF and insulin pathway is highly controversial, and its mechanisms are far from clear. This study looked into the relationship between PBEF, insulin receptor (IR) and insulin pathway from a new perspective-lipid rafts, which revealed a potential mechanism for inflammation induced insulin resistance.Methods:Detergent and detergent-free methods were used to isolate lipid rafts from A549cells, and the distribution of PBEF and IRβ was tested. Recombined PBEF plasmids and its mutant S199were constructed, and PBEF and S199proteins were purified. Cells were given extracellular treatment to study the effect of PBEF on the translocalition of IRβ. Immunoprecipitation (IP) was performed to study the interaction between PBEF, IRβ and Cav-1, as well as phosphorylated Cav-1. The effect of PBEF on insulin-induced Akt activation at different time points was also studied.Results:PBEF was found in both of lipid rafts and non-rafts, while IRP was only found in lipid rafts in detergent-free method. PBEF could induce the movement of IRβ from lipid rafts to non-rafts. NAD had similar effect. This effect could not be blocked by S199or FK866. IRβ, PBEF and Cav-1bound with each other. The tyrosine phosphorylation level of the part of Cav-1that binding with IRβ in non-rafts was higher after PBEF treatment. When adding PBEF within5min following insulin treatment, Akt phosphorylation was inhibited comparing with only giving insulin. However, when adding PBEF after5min following insulin treatment, this effect was partly compromised.Conclusions:1. PBEF was located in both of lipid rafts and non-rafts. PBEF could induce the movement of IRβ from lipid rafts to non-rafts. This effect was partly dependent on its Nampt activity.2. IRβ, PBEF and Cav-lbound with each other. PBEF could induce the co-movement of phosphorylated Cav-1with IRβ into non-rafts, which was dependent on its Nampt activity.3. PBEF could affect insulin-induced Akt phosphorylation, which was time dependent.更多还原